Chronic proliferative diseases are often accompanied by profound angiogenesis, which can contribute to or maintain an inflammatory and/or proliferative state, or which leads to tissue destruction through the invasive proliferation of blood vessels. (Folkman, EXS 79:1–8, 1997; Folkman, Nature Medicine 1:27–31, 1995; Folkman and Shing, J. Biol. Chem. 267:10931, 1992).
Angiogenesis is generally used to describe the development of new or replacement blood vessels, or neovascularisation. It is a necessary and physiological normal process by which the vasculature is established in the embryo. Angiogenesis does not occur, in general, in most normal adult tissues, exceptions being sites of ovulation, menses and wound healing. Many diseases, however, are characterized by persistent and unregulated angiogenesis. For instance, in arthritis, new capillary blood vessels invade the joint and destroy cartilage (Colville-Nash and Scott, Ann. Rheum. Dis., 51, 919,1992). In diabetes (and in many different eye diseases), new vessels invade the macula or retina or other ocular structures, and may cause blindness (Brooks et al., Cell, 79, 1157, 1994). The process of atherosclerosis has been linked to angiogenesis (Kahlon et al., Can. J. Cardiol. 8, 60, 1992). Tumor growth and metastasis have been found to be angiogenesis-dependent (Folkman, Cancer Biol, 3, 65, 1992; Denekamp, Br. J. Rad. 66, 181, 1993; Fidler and Ellis, Cell, 79, 185, 1994).
The recognition of the involvement of angiogenesis in major diseases has been accompanied by research to identify and develop inhibitors of angiogenesis. These inhibitors are generally classified in response to discrete targets in the angiogenesis cascade, such as activation of endothelial cells by an angiogenic signal; synthesis and release of degradative enzymes; endothelial cell migration; proliferation of endothelial cells; and formation of capillary tubules. Therefore, angiogenesis occurs in many stages and attempts are underway to discover and develop compounds that work to block angiogenesis at these various stages.
There are publications that teach that inhibitors of angiogenesis, working by diverse mechanisms, are beneficial in diseases such as cancer and metastasis (O'Reilly et al., Cell, 79, 315, 1994; Ingber et al., Nature, 348, 555, 1990), ocular diseases (Friedlander et al., Science, 270, 1500, 1995), arthritis (Peacock et al., J. Exp. Med. 175, 1135, 1992; Peacock et al., Cell. Immun. 160, 178, 1995) and hemangioma (Taraboletti et al., J. Natl. Cancer Inst. 87, 293, 1995).
Angiogenesis signals result from the interaction of specific ligands with their receptors. The Tie1 and Tie2 receptors are single-transmembrane, tyrosine kinase receptors (Tie stands for Tyosine kinase receptors with immunoglobulin and EGF homology domains). Both were recently cloned and reported by several groups (Dumont et al., Oncogene 8:1293–1301, 1993; Partanen et al., Mol. Cell Biol. 12:1698–1707, 1992; Sato et al., Proc. Natl. Acad. Sci. USA 90:9355–9358, 1993).
The Tie receptors are proteins of approximately 125 kDa, with a single putative transmembrane region. The extracellular domain of these receptors is divided into several regions: there are 3 regions that have a pattern of cysteine expression found in EGF-like domains; there are 2 regions that have some weak homology to and structural characteristics of immunoglobulin-like domains; and there are 3 regions with homology to the fibronectin III repeat structure. This particular combination of extracellular domains is unique to the Tie receptors. The intracellular portion of Tie2 is most closely related (˜40% identity) to the kinase domains of FGF-R1, PDGF-R and c-kit. The intracellular portions of Tie2 contain all of the features of tyrosine kinases, including a GXGXXG ATP binding site consensus sequence and typical tyrosine kinase motifs (i.e., HRDLAARN and DFGL).
These receptors have sparked interest because they are the only receptor tyrosine kinases, other than those receptors for vascular endothelial cell growth factor (VEGF), that are largely restricted to endothelial cells in their expression. There are several lines of evidence showing that Tie2 is important in angiogenesis, detailed in the following paragraphs.
a. Tie1 and Tie2 Receptor Location
i. Embryological Vascular Development
The location of the Tie receptors in the embryo has been studied by a number of investigators using in situ hybridization. Korhonen et al. (Blood 80:2548–2555, 1992) showed that the mRNA for Tie receptors is located in endothelial cells of all forming blood vessels and in the endocardium of mouse embryos. During embryonic development, expression of the Tie receptors is seen in angioblasts and all developing vasculature. Expression of the Tie receptors follows expression of the major VEGF receptor, Flk-1, by 12–24 hours during mouse embryogenesis, perhaps suggesting sequential and different actions of these receptor systems (Schnurch and Risau, Development 119: 957–968, 1993). Cloning of a 1.2 Kb genomic 5′ flanking region of Tie2, coupled to a lacZ gene and expressed in transgenic mice, demonstrated a selective pattern of expression in endothelial cells during embryonic development (Schlaeger et al., Development 121:1089–1098, 1995). Thus, the Tie2 promoter acts to assure endothelial cell-specific expression of Tie2.
ii. In Adult Tissues
The similarities between embryonic angiogenesis and pathologic angiogenesis yields the hypothesis that blocking Tie2 function, in tumors or chronic inflammatory settings, for examples, may block angiogenesis, thus blocking further cell proliferation and provide therapeutic benefit. Tie mRNA cannot be observed in adult skin, except at sites of active wound healing, where the proliferating capillaries in the granulation tissue contain abundant Tie mRNA (Korhonen et al., Blood 80:2548–2555, 1992). PCR amplification of cDNA from normal skin fails to show a signal for Tie receptor (Kaipainen et al., Cancer Res. 54:6571–6577, 1994). In contrast, a strong signal is seen with cDNA from metastasizing melanomas, where in situ studies localize this signal to the vascular endothelium. While Tie receptor expression is down-regulated in the established vasculature, it is up-regulated in the angiogenesis that occurs in the ovary during ovulation, in wounds and in tumor (breast, melanoma and renal cell carcinoma) vasculature, consistent with prevailing views that angiogenesis in the adult borrows from embryonic angiogenic mechanisms.
b. Tie Knockout Animals
Homozygous mice with a Tie2 knockout, or carrying a transgene encoding a “dominant-negative” Tie2 receptor, confirmed that the Tie2 receptor is critical for embryonic development (Dumont et al., Genes Dev. 8:1897–1909, 1994; Sato et al., Nature 376:70–74, 1995). Embryonic death in these mice occurred due to vascular insufficiency and there were dramatically reduced numbers of endothelial cells. Vasculogenesis—that is the differentiation of endothelial cells and the in situ formation of vessels—appeared relatively normal in mice lacking Tie2. The subsequent sprouting and remodeling resulting in formation of vessel branches (angiogenesis) was drastically reduced in the Tie2 mutant mice embryos. This lack of sprouting and angiogenesis resulted in substantial growth retardation, particularly of the brain, neural tube and heart, resulting in lack of viability. This exemplifies the critical importance of Tie2 in angiogenesis. This is significant, as angiogenesis is regulated by a number of growth factors. Interestingly, Flk1 (VEGF receptor) knockout mice exhibit embryo lethal defects in vasculogenesis, that occur earlier than those of Tie2 disruption. Disruption of the Tie1 receptor yields a much different, and later, defective phenotype; the mouse embryo dies late in development due to hemorrhage resulting from defective integrity of an otherwise well-formed vasculature. Taken together, these studies suggest that the VEGF/Flk1 and Tie systems operate in sequential fashion, with Tie2 having a critical role in angiogenesis.
c. Tie2 Ligands
Recently, two ligands for the Tie2 receptor have been reported. Angiopoietin-1 binds and induces the tyrosine phosphorylation of Tie2 and its expression in vivo is in close proximity with developing blood vessels (Davis et al., Cell 87:1161–1169, 1996). Mice engineered to lack Angiopoietin-1 display angiogenic deficits reminiscent of those previously seen in mice lacking Tie2 receptors, demonstrating that Angiopoietin-1 is a primary physiologic ligand for Tie2 and that Tie2 has critical in vivo angiogenic actions (Suri et al., Cell 87:1171–1180, 1996). Angiopoietin-2 was identified by homology screening and shown to be a naturally occurring antagonist for Tie2 receptors. Transgenic overexpression of Angiopoietin-2 disrupts blood vessel formation in the mouse embryo (Maisonpierre et al., Science 277:55–60, 1997). Together, these results support a role for Tie2 receptors in angiogenesis.
d. Tie2 Inhibition
Based upon the importance of Tie2 receptors in angiogenesis, inhibition of Tie2 kinase activity is predicted to interrupt angiogenesis, providing disease-specific therapeutic effects. Recently, Lin et al. (J. Clin. Invest. 100:2072–2078, 1997) has shown that exogenously administered soluble Tie2 receptor inhibited angiogenesis and cancer growth in animal models. Thus inhibition of Tie2 receptors by other means, such as inhibition of Tie2 receptor kinase activity, is expected to have therapeutic benefit in proliferative diseases involving angiogenesis.
The current application teaches the novel finding that compounds of specific structure can inhibit the kinase activity of the Tie2 receptor, block its signal transduction and thus may be beneficial for proliferative diseases via inhibition of signals for angiogenesis.